Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that permit thrombin and other extracellular proteases to regulate cellular behaviors. Together with the coagulation cascade, PARs link tissue injury to cellular responses that help orchestrate hemostasis and thrombosis, inflammation, cytoprotection, repair, and pain perception. Given these and other roles, PARs are potential drug targets. Available evidence supports a model in which thrombin activates the prototypical PAR, PAR1, by cleaving the N-terminal exodomain of the receptor at a specific site to generate a new N-terminus that then functions as a tethered peptide agonist, binding intramolecularly to the receptor's heptahelical bundle to effect receptor activation. Structures that test this model and reveal how the tethered ligand binds and how such binding drives transmembrane domain (TM) movement and G protein activation are lacking, as are structures to support development of pharmaceuticals targeting PARs. Building on our recent crystal structure of inactive-state PAR1 complexed with the antagonist vorapaxar, we propose studies to illuminate the mechanism of PAR activation, signaling and antagonism at a structural level. We will 1) Solve crystal structures of thrombin- activated PAR1 in complex with Gi and either Gq or G12/13. 2) Determine the basis for vorapaxar's specificity for PAR1 over closely related receptors and the route of vorapaxar entry (from the plasma membrane or the extracellular space), and 3) Solve the crystal structure of a PAR2-antagonist complex. Cutting edge crystallographic approaches, including use of stabilizing nanobodies and single particle EM to assess complexes, will be employed. Molecular dynamics simulations will aid design and interpretation of mutational studies. Our studies of PAR1 will reveal the mechanism by which the PAR1 tethered agonist binds and triggers TM movement and G protein activation, the structural basis for PAR1's promiscuous coupling to multiple G protein subtypes (Gi, Gq, and G12/13), a novel route of antagonist entry and the importance of entry route for specificity. Detailed studies of the PAR1-vorapaxar structure and the PAR2 crystal structure will provide an entry to structure-based discovery and optimization of better PAR antagonists needed to explore the role of these receptors in human disease. Our studies will provide the first structure of a peptide agonist- GPCR-G protein complex and the first structural insight into whether distinct conformers of individual GPCRs recognize different G proteins.